A pluggable, fluxable multi-organ interconnection chip

Abstract

The application relates to a pluggable and fluxable multi-organ interconnection chip, which comprises a cover plate, a plurality of organ culture chambers, a multi-organ interconnection chip base and a high-transparency bottom sealing film attached to the bottom of the multi-organ interconnection chip base; the multi-organ interconnection chip base is provided with a plurality of chamber insertion holes, an inlet and an outlet for perfusion of culture solution, micro-pipes connected with the organs, and two, four or six connected chambers; the chamber insertion holes are provided with the plurality of organ culture chambers which can be embedded into the multi-organ interconnection chip base; and the cover plate is installed on the upper part of the multi-organ interconnection chip base and is used for preventing pollution. The application can realize multi-organ interconnection and perfusion culture, has the advantages of simple production, easy batch production, strong compatibility, compatibility with multiple organ culture, microscopic observation, compatibility with multiple commercial detection devices, compatibility with automatic devices, chamber plugging and unplugging according to certain procedures, and meeting the requirements of modern drug research and development industry automatic operation.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of bio-device manufacturing, in particular to a plug-in and fluxing multi-organ interconnection chip. BACKGROUND

[0002] Drug development is an inefficient resource-intensive process, with the average cost of new drug development often exceeding 1 billion dollars. Many drugs fail in the third phase of testing or show serious side effects after being marketed, ultimately leading to the failure of drug development. The limited predictive ability of animal models is the main reason for the failure of 90% of candidate drugs in human trials. Current medical research mainly relies on simple human cell culture or rodent models. Simple cell culture is intuitive and easy to operate, but its predictive ability is limited due to the lack of biological functions. In contrast, animal models have more powerful physiological functions, but due to the differences between animal and human physiology, they result in prediction failures. Organ chip technology seeks to combine the advantages of the two models by culturing human cells in a tissue-specific three-dimensional environment, simulating complex human systems by constructing human genetic, physiological, and pathological diversity characteristics, which can be used for medical research and improving drug development efficiency.

[0003] Single-organ chips focus on mimicking the function of a single organ, but simulating the interaction between organs in higher-level organizations remains very challenging. From a holistic perspective, the complexity of the human body arises from the dynamic interaction between its constituent parts at different levels within the organization, and simulating these interactions is crucial to mimicking the integrated behavior of complex physiological systems in vitro. Multi-organ chips, which integrate multiple organ units into a whole, are connected by pipes between different organs to form systems similar to the human digestive, motor, and immune systems. Therefore, the development of a multi-organ chip with strong compatibility and fluxing has great application prospects in drug development and personalized medicine. SUMMARY

[0004] The technical problem to be solved by the present application is to provide a plug-in and fluxing multi-organ interconnection chip, which can be used as a multi-cell culture, organ culture, and multi-organ co-culture device for drug screening and personalized medicine.

[0005] The technical solution adopted by the present application to solve the technical problem is to provide a plug-in and fluxing multi-organ interconnection chip, which comprises a cover plate on a multi-organ interconnection chip base, a plurality of organ culture chamber inserts on the multi-organ interconnection chip base, and a high-transparency bottom sealing film attached to the bottom of the multi-organ interconnection chip base.

[0006] The cover plate and the chip base can be formed by mechanical carving, laser carving, three-dimensional printing, casting, and other processing methods.

[0007] The cover plate is embedded on the upper part of the chip base, and the cover plate can be loaded or removed by various methods such as manual or negative pressure suction plate mechanical arm.

[0008] The cover plate is colorless and transparent, and the organ culture state in the culture chip can be observed in real time through the cover plate.

[0009] The plurality of organ culture chamber inserts are processed Transwell chamber analogs or directly use commercial Transwell chamber inserts;

[0010] The plurality of organ culture chamber inserts are composed of a side wall and a bottom porous structure, and the porous structure can be a microchannel, a porous membrane, a micro fence, or a hydrogel, etc. with porous permeability.

[0011] The plurality of organ culture chamber inserts are connected with the multi-organ interconnection base through a microchannel, a porous membrane, a micro fence, or a hydrogel. The presence of the microchannel, the porous membrane, the micro fence, or the hydrogel avoids the cells in the cell culture chamber from entering the interconnection base or the cells in the base from entering the organ culture chamber insert, while allowing liquid, cytokines, exosomes, drugs, or cell metabolites to pass through, realizing interconnection communication between organs.

[0012] Further, at least one positioning column is arranged on the outer side wall surface of the organ culture chamber insert, and the cell culture chamber insert is clamped into the multi-organ chip base by various methods such as bag rotation and embedding. Therefore, according to the needs, the fluid perfusion can realize the arbitrary interconnection between different organs.

[0013] Further, at least one mechanical hand clamping position is arranged on the top of the outer side wall surface of the organ culture chamber insert, and the mechanical hand clamping position can be of any shape, facilitating the mechanical hand to grasp the organ culture chamber insert and accurately clamp it into the chamber insert hole of the chip base.

[0014] Each organ culture chamber insert can culture one or more organs, and the organs can exist in the form of cell balls, organoids, living tissues, etc. in the interior or exterior of the chamber insert. The culture method of the cells includes adhering to the bottom porous structure of the chamber insert or suspending in the liquid culture medium or culturing in the three-dimensional matrix.

[0015] The chip base has various structures such as an external liquid access hole, a fluid access chip base channel, an internal fluid perfusion hole, a chamber insert hole, a chamber insert bayonet, an organ communication flow channel, and a fluid outflow chip channel.

[0016] The size specification of the chip substrate is the same as that of a commercial cell culture 6-well plate, 12-well plate, 24-well plate, 48-well plate, 96-well plate, and is compatible with various detection equipment such as an enzyme label instrument and a microscope; the chamber insertion hole position is consistent with the center position of a culture hole of a commercial well plate, and is compatible with various automatic pipetting devices.

[0017] The external connection hole of the chip substrate is connected to an external diaphragm pump, peristaltic pump, air pressure pump or syringe pump through a luer adapter and the like to pump the culture medium into the chip.

[0018] The chip substrate has at least two fluid perfusion channels, one of which is a fluid inlet channel of the chip substrate, and the other of which is a fluid outlet channel of the chip substrate, and the two liquid perfusion channel channels are respectively embedded in the long axis direction of the chip substrate and connected to the external liquid connection hole at both ends.

[0019] The fluid inlet channel of the chip substrate is slightly higher than the fluid inlet and outlet channel of the chip substrate in the horizontal height, and the height difference of the liquid level inside the chip substrate can drive the fluid flow.

[0020] The chip substrate has a plurality of organ culture chamber insertion holes inside, which form different arrays according to the number of co-cultured organs and have scalability.

[0021] The organ culture chamber insertion hole near the fluid perfusion channel or on both sides has an arc bevel which is tangent or intersects with the fluid perfusion channel, forming a plurality of internal fluid perfusion holes.

[0022] The organ culture chamber insertion hole side has a symmetrical chamber insertion bayonet, and the organ culture chamber insertion can be vertically inserted into the substrate, rotated by a certain angle, and fixed in the chip substrate to facilitate culture or observation.

[0023] The organ culture chamber insertion holes are connected to each other through the organ communication flow channel at the bottom of the chip substrate, allowing any connection between two or more same or different organs, providing a blood-like connection between the organs through fluid perfusion, and providing a shear force environment for the organs.

[0024] The bottom of the chip substrate is sealed by a sealing film, which can be a high-transparency sealing film or glass or a high-molecular-weight light-transmitting material, to seal the channels at the bottom of the chip substrate. While providing a closed liquid environment, it also provides high light transmission for microscopic observation of cells or organs.

[0025] The application provides a use mode of a multi-organ interconnection culture chip that can be plugged in, which includes:

[0026] Vascular endothelial cells are inoculated outside the porous structure at the bottom of the organ insertion chamber, and cultured at 37°C to obtain a chip with vascular communication structure.

[0027] Inoculate the cell suspension or cell cluster or organoid or microtissue mixed with different organ cells and matrix material outside the porous structure at the bottom of the organ insert chamber, and culture at 37 DEG C to obtain a chamber insert of various organs;

[0028] The organ insert containing the vascular cell layer is arranged in the micro-chamber of the chip in sequence according to experimental requirements, and the bottom of the chip substrate is closed by using a high-transmittance membrane.

[0029] The pluggable multi-organ interconnected culture chip is connected with external pipelines and external pump devices, perfusion culture is carried out in a 37 DEG C incubator or other small cell culture devices using vascular endothelial cell culture medium.

[0030] A construction method of a barrier or spherical organoid, the specific steps are as follows:

[0031] Step one: collagen, including I, II, III, IV, V, VI and other collagens, matrix glue or hydrogel and other materials with coating function, is used to coat the inside or outside of the porous material at the bottom of the organ culture chamber.

[0032] Step two: a certain concentration of vascular endothelial cell suspension is added to the outside of the porous structure at the bottom of the organ culture chamber according to a certain volume, and is allowed to stand for at least 2 hours to allow the cells to adhere and grow to form blood vessels.

[0033] Step three: a certain concentration of barrier organoid cell suspension such as intestinal, skin, lung, blood-brain barrier, etc. is added to the inside of the porous structure at the bottom of the organ culture chamber according to a certain volume, and the required culture medium for the organ culture is added, and is allowed to stand for at least 2 hours to allow the cells to adhere and grow to form a barrier organoid containing blood vessels.

[0034] Step four: a certain concentration of spherical organoid mixed matrix glue suspension such as liver, heart or other organoids is added to the inside of the porous structure at the bottom of the organ culture chamber according to a certain volume, and after the matrix glue is heated and solidified in the incubator, the required culture medium for the organ culture is added for standing or perfusion culture.

[0035] Beneficial effects: the present application relates to a pluggable and fluxable multi-organ interconnected chip, which has the following advantages:

[0036] (1) The present application provides a multi-organ co-culture chip, which is simple to make as a whole, can be perfusion cultured, has a size consistent with a commercial cell culture multi-well plate as a whole, is compatible with various culture or detection equipment, and can be plugged and unplugged in a certain program by an automatic device to meet the automatic operation requirements of modern drug research and development industry.

[0037] (2), the chip provided by the application can carry out various forms of organ culture, and can construct organs according to physiological characteristics of different organs; meanwhile, various culture chambers are relatively independent, and can be independently cultured by using culture medium required by organs;

[0038] (3), the multiple organ connection mode provided by the application is through a channel at the bottom of the chip, the chip can be cultured by circulation perfusion by using external pipelines or pump devices, meanwhile, the channel has intravascular endothelial cells and a height difference, and the organ perfusion of the blood circulation system can be simulated in the chip;

[0039] (4), the created co-culture interconnection chip can assemble any multiple organs according to requirements, the number of co-cultured organs has expandability, and two or more organs can be connected and co-cultured according to requirements. BRIEF DESCRIPTION OF DRAWINGS

[0040] Figure 1 is an exploded schematic view of the plug-in multiple organ interconnection culture chip described in the application;

[0041] Figure 2 is an exploded schematic view of the organ culture chamber insert described in the application;

[0042] Figure 3 is a top view of the plug-in multiple organ culture chip described in the application;

[0043] Figure 4 is a side view of the plug-in multiple organ interconnection culture chip described in the application;

[0044] Figure 5 is a bright field photo of the small intestine barrier organoid described in the application.

[0045] Fig. 1, cover plate, 2, multiple organ culture chamber insert, 3, multiple organ interconnection chip base, 4, high-transmittance bottom sealing film, 2-1, mechanical hand clamping position, 2-2, positioning column, 2-3, porous structure, 3-1, external connection hole, 3-2, fluid inlet chip base channel, 3-3, internal fluid perfusion hole, 3-4, chamber insertion hole, 3-5, chamber insertion bayonet, 3-6, organ communication flow channel, 3-7, fluid outflow chip channel. DETAILED DESCRIPTION

[0046] The application will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the application and not used to limit the scope of the application. In addition, it should be understood that those skilled in the art can make various modifications or changes to the application after reading the content of the application, and these equivalent forms also fall within the scope of the appended claims of the application.

[0047] Embodiments of the present application relate to a pluggable, fluxable multi-organ interconnection chip, such as Figure 1 The pluggable multi-organ interconnection culture chip shown in FIG. 4 comprises a cover plate 1, a plurality of organ culture chamber inserts 2, a multi-organ interconnection chip substrate 3, and a high-transparency bottom sealing film 4 attached to the bottom of the multi-organ interconnection chip substrate.

[0048] The cover plate 1 and the multi-organ interconnection chip substrate 3 can be formed by mechanical carving, laser carving, three-dimensional printing, casting, and other processing methods. Polymethyl methacrylate (PMMA), polystyrene (PS), or some three-dimensional printing materials with high biocompatibility can be used. The size of the multi-organ interconnection chip substrate 3 is consistent with that of a commercial multi-well cell culture plate, with a long axis length of 127.50 mm and a short axis length of 82.25 mm.

[0049] The cover plate 1 is embedded in the upper part of the multi-organ interconnection chip substrate 3 and can be placed or removed by manual or negative pressure suction cup and other methods.

[0050] The cover plate 1 is colorless and transparent, allowing real-time observation of the organ culture state in the culture chip.

[0051] The plurality of organ culture chamber inserts 2 are processed Transwell chamber analogs or directly use commercial Transwell chamber inserts.

[0052] The plurality of organ culture chamber inserts 2 are composed of a side wall and a bottom porous structure 2-3. The porous structure 2-3 can be a microchannel, a porous membrane, a micro-lattice, or a hydrogel with porous permeability.

[0053] Further, at least one positioning column 2-2 is provided on the outer wall surface of the organ culture chamber insert 2. Preferably, two cuboid or cylindrical positioning columns 2-2 are symmetrically distributed on the outer side wall of the organ culture chamber, with a protruding height of 0.5-3 mm. The cell culture chamber insert is clamped into the multi-organ chip substrate by embedding, rotating, and other methods, and the positioning column realizes precise positioning of the organ culture chamber insert 2. Therefore, different organs can be interconnected as needed through fluid perfusion.

[0054] Further, at least one mechanical hand clamping position 2-1 is arranged on the top of the outer wall of the organ culture chamber insert 2. The mechanical hand clamping position can be of any shape, which is convenient for the mechanical hand to grasp the organ culture chamber insert 2 and accurately clamp it into the chip base chamber insertion hole 3-4. Preferably, two pincer-shaped mechanical hand clamping positions 2-1 are symmetrically arranged on the top of the outer wall of the organ culture chamber, and the length is 2-10 mm.

[0055] Each organ culture chamber insert 2 can culture one or more organs. The organs can exist in the form of cell aggregates such as adherent culture, cell spheres, organoids, and living tissues inside or outside the chamber insert. The culture method of cells includes adhering to the porous structure 2-4 of the chamber insert or suspending in the liquid culture medium or culturing in the three-dimensional matrix.

[0056] The multi-organ interconnected chip base 3 has various structures such as external liquid connection hole 3-1, fluid inlet chip base channel 3-2, internal fluid perfusion hole 3-3, chamber insertion hole 3-4, chamber insertion bayonet 3-5, organ communication flow channel 3-6, and fluid outlet chip channel 3-7.

[0057] The external connection hole 3-1 of the multi-organ interconnected chip base 3 is connected to an external diaphragm pump, a peristaltic pump, an air pressure pump, or a syringe pump through a micro-pipe such as a luer connector to pump the culture medium into the chip.

[0058] The multi-organ interconnected chip base 3 has at least two fluid perfusion channels, one of which is the fluid inlet chip base channel 3-2, and the other of which is the fluid outlet chip channel 3-7. The fluid channel has a diameter of 1-5 mm. Taking a four-organ co-culture chip as an example, two liquid perfusion flow channels 3-2 are embedded in the long axis direction of the multi-organ interconnected chip base 3, 4-10 mm away from the bottom of the chip base, and the two ends are connected to the external liquid connection hole 3-1 in the vertical direction. The external hole has a diameter of 2-5 mm and is used in cooperation with a commercial fluid component luer connector.

[0059] The fluid inlet chip base channel 3-2 is slightly higher than the fluid inlet and outlet chip base channel 3-7 in horizontal height, 0.5-3 mm, and the height difference of the liquid level inside the chip base can drive the flow of fluid.

[0060] The chip base has a plurality of organ culture chamber insertion holes 3-4 inside. According to the number of co-cultured organs, different arrays are formed, which have scalability. Taking a four-organ co-culture chip as an example, the organ culture chamber insertion holes are arranged in 4x6 in the multi-organ interconnected chip base 3.

[0061] The two sides of the organ culture chamber insertion hole 3-4 close to the fluid perfusion channel 3-2 or the fluid outflow channel 3-7 of the chip have a circular arc slope, which intersects with the fluid perfusion channel 3-2 or the fluid outflow channel 3-7 of the chip, forming six internal fluid perfusion holes 3-3 on each side.

[0062] The side of the organ culture chamber insertion hole 3-4 has a symmetric chamber insertion bayonet 3-5, and the organ culture chamber insertion can be fixed in the chip substrate after being vertically inserted into the substrate and rotated by an angle of 1-90°, which is convenient for culture or observation.

[0063] The organ culture chamber insertion hole 3-4 is connected by the organ communication flow channel 3-6 at the bottom of the multi-organ interconnection chip substrate 3, allowing two or more than two same or different organs to be connected arbitrarily, providing a blood-like connection between organs through fluid perfusion, and providing a shear force environment for the organs;

[0064] The high-transmittance sealing film 4 can be a high-transmittance sealing film or a glass or a high-molecular transmittance material, which can provide a high-transmittance for microscopic observation of cells or organs while providing a closed liquid environment.

[0065] Embodiment 1

[0066] The organ insertion chamber 2 is inoculated with vascular endothelial cells on the outside of the porous structure 2-2 at the bottom of the chamber, and cultured at 37°C to obtain a chip with vascular communication structure;

[0067] The organ insertion chamber 2 is inoculated with a mixture of different organ cells and matrix material, cell suspension or cell mass or organoid or microtissue on the inside of the porous structure 2-2 at the bottom of the chamber, and cultured at 37°C to obtain a multi-organ chamber insertion;

[0068] The removable multi-organ interconnection culture chip is sterilized by high pressure or ultraviolet or alcohol immersion, and the bottom channel is sealed with a high-transmittance sealing film 4.

[0069] Before multi-organ co-culture, incubate with complete medium for at least 30 minutes to construct a suitable culture environment.

[0070] The above different organ insert containing vascular cell layer is arranged in the chip culture micro-pore according to the experimental requirements.

[0071] The fluid external connection hole 3-1 on the chip substrate 2 is connected to the external diaphragm pump device through a luer connector and a pipeline, and the vascular endothelial cell culture medium is used for perfusion culture at a fluid speed of 0.3-1.5 mL / min in a 37°C incubator or other small cell culture device, and the medium in the organ culture chamber insertion is replaced every 24-48 hours.

[0072] Example 2

[0073] (1) Collagen I PBS solution with concentration of 50-500 μg / mL is prepared, 10-200 μL of which is added to each organ culture chamber to coat the bottom porous material of the organ culture chamber at a density of 5-20 μg / m2, and the organ culture chamber is incubated at 4°C overnight.

[0074] (2) The next day, the organ culture chamber insert is washed 1-3 times with clean PBS. The organ culture chamber insert is inverted, and 10-200 μL of Matrigel diluted with PBS to a concentration of 5-30% and 10-200 μL of the coating solution diluted with PBS to a concentration of 5-30% are added to the bottom porous structure (2-2) of each organ culture chamber insert, and the organ culture chamber is incubated at room temperature for 1 h and then washed once with clean PBS.

[0075] (3) 50-200 μL of a suspension of vascular endothelial cells at a concentration of 0.1-5 x 10 6 cells / mL is added to the outside of the bottom porous structure 2-2 of the organ culture chamber, and the organ culture chamber is incubated at 37°C for at least 2 h to allow the cells to adhere and grow.

[0076] (4) The organ culture chamber insert is inverted, and a suspension of Caco-2 cells at a concentration of 0.1-5 x 10 6 cells / mL is prepared, 50-200 μL of which is added to the non-vascular side of the bottom porous structure of each organ culture chamber, and sufficient culture medium is added, and the organ culture chamber is incubated at 37°C for at least 2 h to allow the cells to adhere and grow to form a small intestine barrier organoid containing blood vessels, as shown in Figure 5 .

[0077] The above provides a pluggable and fluxed multi-organ interconnection chip, and the principles and implementation manners of the present application are described by using specific examples in the present application. The above description of the examples is only used to help understand the method and core idea of the present application; meanwhile, for those skilled in the art, the specific implementation manners and application ranges can be changed according to the idea of the present application, and the above description of the present application should not be understood as a limitation.

Claims

1. A pluggable, fluxed, multi-organ interconnection chip, characterized by: The application relates to a multi-organ interconnection chip, which comprises a cover plate (1), a plurality of organ culture chamber inserts (2), a multi-organ interconnection chip base (3) and a high-transmittance bottom sealing film (4) attached to the bottom of the multi-organ interconnection chip base (3). The multi-organ interconnection chip base (3) is provided with a plurality of chamber insertion holes (3-4), the chamber insertion holes (3-4) are provided with the plurality of organ culture chamber inserts (2), the cover plate (1) is arranged on the upper part of the multi-organ interconnection chip base (3) and is used for sealing the multi-organ interconnection chip base (3), the multi-organ interconnection chip base (3) is provided with a plurality of organ communication flow channels (3-6) which are communicated with the bottoms of the chamber insertion holes (3-4), the multi-organ interconnection chip base (3) is provided with at least two fluid perfusion channels, one of which is a fluid inlet chip base channel (3-2) and the other is a fluid outlet chip channel (3-7), the chamber insertion holes (3-4) are provided with internal fluid perfusion holes (3-3) which are communicated with the fluid inlet chip base channel (3-2) or the fluid outlet chip channel (3-7), the multi-organ interconnection chip base (3) is provided with an external connecting hole (3-1) which is communicated with the fluid inlet chip base channel (3-2), the fluid inlet chip base channel (3-2) is higher than the fluid outlet chip channel (3-7) by 0.5-3 mm in the horizontal height, the fluid flow is driven by the height difference of the liquid level in the chip base, the chamber insertion holes (3-4) are provided with symmetrical chamber insertion bayonets (3-5) on the side surfaces, the plurality of organ culture chamber inserts (2) are vertically inserted, then are rotated by an angle of 1-90 DEG, so that the plurality of organ culture chamber inserts (2) are fixed in the multi-organ interconnection chip base (3), the chamber insertion holes (3-4) near the fluid perfusion channels or the fluid outlet chip channel (3-7) are provided with a circular arc inclined surface which is intersected with the fluid perfusion channels or the fluid outlet chip channel (3-7) and forms six internal fluid perfusion holes (3-3) on each side.

2. The pluggable, fluxed, multi-organ interconnected chip of claim 1, wherein: The bottom of the plurality of organ culture chamber inserts (2) is provided with a porous structure (2-3) which is made of a porous permeable material.

3. The pluggable, fluxed, multi-organ interconnected chip of claim 1, wherein: The outer side wall of the plurality of organ culture chamber inserts (2) is provided with at least one positioning column (2-2).

4. The pluggable, fluxed, multi-organ interconnected chip of claim 1, wherein: The top of the outer side wall of the plurality of organ culture chamber inserts (2) is provided with at least one mechanical hand clamping position (2-1).

5. The pluggable, fluxed, multi-organ interconnected chip of claim 4, wherein: The mechanical hand clamping position (2-1) is in a pincer-shaped structure and has two symmetrical positioning columns which are distributed on the plurality of organ culture chamber inserts (2) and have a length of 2-10 mm.

6. The multi-organ interconnection chip which can be inserted and has flux according to claim 1, wherein the high-transmittance bottom sealing film (4) is made of glass or a high-molecular transmittance material. ​

Images